Comparative Hepatology - Latest Articles

Bile constituents in hibernating golden-mantled ground squirrels (Spermophilus lateralis)

Julie Baker — 2009-05-26T00:00:00Z

Background: Golden-mantled ground squirrels (S. lateralis) are anorexic during the winter and survive by exploiting hibernation to reduce energetic demands. The liver normally plays a critical role in fueling and regulating metabolism and one might expect significant changes in hepatobiliary function with hibernation. We analyzed bile collected from animals in summer, animals in winter that were either torpid, active between bouts of torpor, or which failed to enter hibernation in order to characterize the effects of hibernation on hepatobiliary function per se. Results: Surprisingly, hibernator bile did not differ from summer squirrel bile in key characteristics including [bile acids], [cholesterol], [free fatty acids], [lecithin], and osmolality. One major distinction between summer and winter squirrels was that winter squirrels experience >5 fold increases in [bilirubin]. Such an increase may have significant physiological consequences that could aid in survivorship of torpor. Animals that failed to hibernate, despite being anorexic, were very similar to summer squirrels in all measured parameters except they had lower bile acid and lecithin concentrations. Conclusion: The data indicate that despite extended anorexia, differences in metabolic fuel privation, and bouts of reduced body temperatures, hibernators normally do not experience broad changes in hepatobiliary function.

Low affinity glucocorticoid binding site ligands as potential anti-fibrogenics

Carylyn Marek — 2009-05-11T00:00:00Z

Background: Pregnane X receptor (PXR) agonists inhibit liver fibrosis. However, the rodent PXR activator pregnenolone 16α carbonitrile (PCN) blocks, in vitro, hepatic stellate cell-to-myofibroblast trans-differentiation and proliferation in cells from mice with a disrupted PXR gene, suggesting there is an additional anti-fibrogenic drug target for PCN. The role of the low affinity glucocorticoid binding site (LAGS) – which may be identical or associated with the progesterone receptor membrane component 1 (PGRMC1) – in mediating this anti-fibrogenic effect has been examined, since binding of dexamethasone to the LAGS in liver microsomal membranes has previously been shown to be inhibited by PCN. Results: Quiescent rat and human hepatic stellate cells (HSC) were isolated from livers and cultured to generate liver myofibroblasts. HSC and myofibroblasts expressed PGRMC1 as determined by RT-PCR and Western blotting. Quiescent rat HSC also expressed the truncated HC5 variant of rPGRMC1. Rat PGRMC1 was cloned and expression in COS-7 cells gave rise to specific binding of radiolabelled dexamethasone in cell extracts that was inhibited by PCN, suggesting that PGRMC1 may be identical to LAGS or activates LAGS binding activity. Liver microsomes were used to screen a range of structurally related compounds for their ability to inhibit radiolabelled dexamethasone binding to rat LAGS. These compounds were also screened for their ability to activate rat and human PXR and to inhibit rat HSC-to-myofibroblast trans-differentiation/proliferation. A compound (4 androstene-3-one 17β-carboxylic acid methyl ester) was identified which bound rat LAGS with high affinity and inhibited both rat and human HSC trans-differentiation/proliferation to fibrogenic myofibroblasts without showing evidence of rat or human PXR agonism. However, despite potent anti-fibrogenic effects in vitro, this compound did not modulate liver fibrosis severity in a rat model of liver fibrosis. Immunohistochemical analysis showed that rat liver myofibroblasts in vivo did not express rPGRMC1. Conclusion: LAGS ligands inhibit HSC trans-differentiation and proliferation in vitro but show little efficacy in inhibiting liver fibrosis, in vivo. The reason(s) for this disparity is/are likely associated with an altered myofibroblast phenotype, in vitro, with expression of rPGMRC1 in vitro but not in vivo. These data emphasize the limitations of in vitro-derived myofibroblasts for predicting their activity in vivo, in studies of fibrogenesis. The data also demonstrate that the anti-fibrogenic effects of PCN in vivo are likely mediated entirely via the PXR.

Phospholipase C beta 4 in mouse hepatocytes: Rhythmic expression and cellular distribution

Brittany Klein — 2008-10-28T00:00:00Z

Background: Circadian regulated physiological processes have been well documented in the mammalian liver. Phospholipases are important mediators of both cytoplasmic and nuclear signaling mechanisms in hepatocytes, and despite a potentially critical role for these enzymes in regulating the temporal aspect of hepatic physiology, their involvement in the circadian liver clock has not been the subject of much investigation. The phospholipase C β4 (PLCβ4) enzyme is of particular interest as it has been linked to circadian clock function. In general, there is no knowledge of the role of the PLCβ4 isozyme in mammalian hepatocytes as this is the first report of its expression in the mammalian liver. Results: We found that in the liver of mice housed on a light:dark cycle, PLCβ4 protein underwent a significant circadian rhythm with a peak occurring during the early night. In constant darkness, the protein rhythm was more robust and peaked around dusk. We also observed a significant oscillation in plcβ4 gene expression in the livers of mice housed in both photoperiodic and constant dark conditions. The cellular distribution of the protein in hepatocytes varied over the course of the circadian day with PLCβ4 primarily cytoplasmic around dusk and nuclear at dawn. Conclusion: Our results indicate that PLCβ4 gene and protein expression is regulated by a circadian clock in the mouse liver and is not dependent on the external photoperiod. A light-independent daily translocation of PLCβ4 implies that it may play a key role in nuclear signaling in hepatocytes and serve as a daily temporal cue for physiological processes in the liver.

Non invasive in vivo investigation of hepatobiliary structure and function in STII medaka (Oryzias latipes): Methodology and applications

Ron Hardman — 2008-10-06T00:00:00Z

Background: A novel transparent stock of medaka (Oryzias latipes; STII), recessive for all pigments found in chromatophores, permits transcutaneous imaging of internal organs and tissues in living individuals. Findings presented describe the development of methodologies for non invasive in vivo investigation in STII medaka, and the successful application of these methodologies to in vivo study of hepatobiliary structure, function, and xenobiotic response, in both 2 and 3 dimensions. Results: Using brightfield, and widefield and confocal fluorescence microscopy, coupled with the in vivo application of fluorescent probes, structural and functional features of the hepatobiliary system, and xenobiotic induced toxicity, were imaged at the cellular level, with high resolution (< 1 μm), in living individuals. The findings presented demonstrate; (1) phenotypic response to xenobiotic exposure can be investigated/imaged in vivo with high resolution (< 1 μm), (2) hepatobiliary transport of solutes from blood to bile can be qualitatively and quantitatively studied/imaged in vivo, (3) hepatobiliary architecture in this lower vertebrate liver can be studied in 3 dimensions, and (4) non invasive in vivo imaging/description of hepatobiliary development in this model can be investigated. Conclusion: The non-invasive in vivo methodologies described are a unique means by which to investigate biological structure, function and xenobiotic response with high resolution in STII medaka. In vivo methodologies also provide the future opportunity to integrate molecular mechanisms (e.g., genomic, proteomic) of disease and toxicity with phenotypic changes at the cellular and system levels of biological organization. While our focus has been the hepatobiliary system, other organ systems are equally amenable to in vivo study, and we consider the potential for discovery, within the context of in vivo investigation in STII medaka, as significant.

A new parameter using serum lactate dehydrogenase and alanine aminotransferase level is useful for predicting the prognosis of patients at an early stage of acute liver injury: a retrospective study

Kazuhiro Kotoh — 2008-08-14T00:00:00Z

Background: Although most patients with severe acute hepatitis are conservatively cured, some progress to acute liver failure (ALF) with a high rate of mortality. Based on the evidence that over-activation of macrophages, followed by disturbance of the hepatic microcirculation, plays a key role in ALF, we hypothesized that the production of serum lactate dehydrogenase (LDH) might increase in the liver under hypoxic conditions and could be an indicator to discriminate between conservative survivors and fatal patients at an early stage. Results: To confirm this hypothesis, we developed a new parameter with serum alanine aminotransferase (ALT) and LDH: the ALT-LDH index = serum ALT/(serum LDH - median of normal LDH range). We analyzed retrospectively 33 patients suffering acute liver injury (serum ALT more than 1000 U/L or prothrombin time expressed as international normalized ratio over 1.5 at admission) and evaluated the prognostic value of the ALT-LDH index, comparing data from the first 5 days of hospitalization with the Model for End-Stage Liver Disease (MELD) score. Patients whose symptoms had appeared more than 10 days before admission were excluded from this study. Among those included, 17 were conservative survivors, 9 underwent liver transplantation (LT) and 7 died waiting for LT. We found a rapid increase in the ALT-LDH index in conservative survivors but not in fatal patients. While the prognostic sensitivity and specificity of the ALT-LDH index was low on admission, at day 3 they were superior to the results of MELD. Conclusion: ALT-LDH index was useful to predict the prognosis of the patients with acute liver injury and should be helpful to begin preparation for LT soon after admission.

Relation between lipogranuloma formation and fibrosis, and the origin of brown pigments in lipogranuloma of the canine liver

Kaori Isobe — 2008-05-12T00:00:00Z

Background: In a previous study we confirmed that canine hepatic lipogranuloma, defined as lesions consisting of small round cells which contain lipid vacuoles and brown pigments in their cytoplasm, was an assembly of Kupffer cells and/or macrophages, and that the cytoplasmic brown pigments in the lesions were hemosiderin and ceroid. However, the pathogenesis of the lesion remains unclear. Kupffer cells (resident macrophages) play a key role in hepatic fibrogenesis due to the production of cytokines including TGF-β. In the present study, we have examined 52 canine liver samples (age: newborn – 14 years; 25 males and 27 females) and investigated the correlation between lipogranuloma formation and fibrosis as well as the origin of brown pigments of lipogranulomas. Results: Lipogranulomas were detected histopathologically in 23 (44.2%) of the 52 liver samples. No significant correlation was found between the density of lipogranulomas and distribution of collagen type I/III in the liver. Pigmentation of lipogranulomas showed significant correlations with that on both hepatocytes and sinusoidal cells, indicating that pigments of lipogranuloma (hemosiderin and ceroid) might be derived from hepatocytes and Kupffer cells. Conclusion: Lipogranulomas are not a contributing factor in hepatic fibrosis, but might be a potential indicator of the accumulation of iron and lipid inside the liver.

The influence of oxygen tension on the structure and function of isolated liver sinusoidal endothelial cells

Inigo Martinez — 2008-05-05T00:00:00Z

Background: Liver sinusoidal endothelial cells (LSECs) are specialized scavenger cells, with crucial roles in maintaining hepatic and systemic homeostasis. Under normal physiological conditions, the oxygen tension encountered in the hepatic sinusoids is in general considerably lower than the oxygen tension in the air; therefore, cultivation of freshly isolated LSECs under more physiologic conditions with regard to oxygen would expect to improve cell survival, structure and function. In this study LSECs were isolated from rats and cultured under either 5% (normoxic) or 20% (hyperoxic) oxygen tensions, and several morpho-functional features were compared. Results: Cultivation of LSECs under normoxia, as opposed to hyperoxia improved the survival of LSECs and scavenger receptor-mediated endocytic activity, reduced the production of the pro-inflammatory mediator, interleukin-6 and increased the production of the anti-inflammatory cytokine, interleukin-10. On the other hand, fenestration, a characteristic feature of LSECs disappeared gradually at the same rate regardless of the oxygen tension. Expression of the cell-adhesion molecule, ICAM-1 at the cell surface was slightly more elevated in cells maintained at hyperoxia. Under normoxia, endogenous generation of hydrogen peroxide was drastically reduced whereas the production of nitric oxide was unaltered. Culture decline in high oxygen-treated cultures was abrogated by administration of catalase, indicating that the toxic effects observed in high oxygen environments is largely caused by endogenous production of hydrogen peroxide. Conclusion: Viability, structure and many of the essential functional characteristics of isolated LSECs are clearly better preserved when the cultures are maintained under more physiologic oxygen levels. Endogenous production of hydrogen peroxide is to a large extent responsible for the toxic effects observed in high oxygen environments.

Sustained virological and biochemical responses to lamivudine and adefovir dipivoxil combination in a chronic hepatitis B infection despite mutations conferring resistance to both drugs

Sylvie Larrat — 2008-03-12T00:00:00Z

Background: Sequential monotherapies of nucleotide analogs used in chronic hepatitis B treatment can lead to the selection of a resistance mutation to each antiviral drug.Case presentationA patient with chronic hepatitis B was successively treated with lamivudine monotherapy, lamivudine-adefovir dual therapy, adefovir monotherapy and again with an adefovir-lamivudine dual therapy. Lamivudine-associated mutations (rtL180M and rtM204V/I) followed by adefovir-associated mutations (rtN236T and rtA181V) emerged during the two monotherapy regimens. Despite the presence of rtM204V/I, rtA181V, and rtN236T mutations at the beginning of the second dual therapy, sustained biochemical and virological responses have been observed thus far after 23 months. Conclusion: This case illustrates that rtM204V/I, rtA181V, and rtN236T resistance mutations can coexist in a patient but do not preclude the recycling of lamivudine and adefovir in combination therapy, when no other therapeutic choices are available.

Over-expression of glutamine synthase in focal nodular hyperplasia (part 1): Early stages in the formation support the hypothesis of a focal hyper-arterialisation with venous (portal and hepatic) and biliary damage

Paulette Bioulac-Sage — 2008-02-29T00:00:00Z

Background: Most focal nodular hyperplasia (FNH) cases are diagnosed by chance. We studied a case of pre-FNH. We used glutamine synthase as an immunohistochemical marker for perivenous zones. Results: Neither fibrotic scars nor hepatocytic nodules surrounded by fibrosis with a ductular reaction were observed in the sections studied. Most sections generally displayed preserved architecture. The glutamine synthase-positive hepatocyte areas were wider than those observed in non-tumoural surrounding liver, and they tended to extend outwards. Portal tracts bordering the nodule were more fibrotic, with an absence of portal veins and ducts and with arterial proliferation often in proximity with large draining veins; isolated arteries were present and hepatic veins were rare in the nodule. These features appeared prior to the identification of other major criteria characteristics of FNH, thus supporting the "hypothesis of Wanless". Conclusion: The findings confirm that in FNH there is a portal tract injury leading to local portal vein injury. This leads to a cascade of events, including arterial venous shunts, ductular reaction, and scar formation.

Plasma redox status is impaired in the portacaval shunted rat - the risk of the reduced antioxidant ability

Maria-Angeles Aller — 2008-02-05T00:00:00Z

Background: Portacaval shunting in rats produces a reduction of hepatic oxidant scavenging ability. Since this imbalance in hepatic oxidant/antioxidant homeostasis could coexist with systemic changes of oxidant stress/antioxidant status, plasma oxidants and antioxidant redox status in plasma of portacaval shunted-rats were determined. Results: Male Wistar male: Control (n = 11) and with portacaval shunt (PCS; n = 11) were used. Plasma levels of the oxidant serum advanced oxidation protein products (AOPP), lipid hydroperoxides (LOOH), the antioxidant total thiol (GSH) and total antioxidant status (TAX) were measured. Albumin, ammonia, Aspartate-aminotransferase (AST), Alanine-aminotransferase (ALT), thiostatin and alpha-1-acid glycoprotein (α1-AGP) were also assayed 4 weeks after the operation. AOPPs were significantly higher (50.51 ± 17.87 vs. 36.25 ± 7.21 μM; p = 0.02) and TAX was significantly lower (0.65 ± 0.03 vs. 0.73 ± 0.06 mM; p = 0.007) in PCS compared to control rats. Also, there was hypoalbuminemia (2.54 ± 0.08 vs. 2.89 ± 0.18 g/dl; p = 0.0001) and hyperammonemia (274.00 ± 92.25 vs. 104.00 ± 48.05 μM; p = 0.0001) and an increase of thiostatin (0.23 ± 0.04 vs. 0.09 ± 0.01 mg/ml; p = 0.001) in rats with a portacaval shunt. The serum concentration of ammonia is correlated with albumin levels (r = 0.624; p = 0.04) and TAX correlates with liver weight (r = 0.729; p = 0.017) and albumin levels (r = 0.79; p = 0.007) Conclusion: These findings suggest that in rats with a portacaval shunt a systemic reduction of oxidant scavenging ability, correlated with hyperammonemia, is principally produced. It could be hypothesized, therefore, that the reduced antioxidant defences would mediate a systemic inflammation.